Alpha/beta hydrolase-fold enzymes

ABSTRACT

The present invention relates to the identification of novel hydrolases in gram positive microorganisms. The present invention provides amino acid sequences for the hydrolase. The present invention provides host cells which comprise nucleic acid encoding the hydrolase. The present invention also provides cleaning compositions, animal feeds and compositions used to treat a textile that include the hydrolase of the present invention.

FIELD OF THE INVENTION

[0001] The present invention relates to alpha/beta hydrolase-foldenzymes derived from gram-positive microorganisms. The present inventionprovides the nucleic acid and amino acid sequences for the hydrolasesand methods for their use.

BACKGROUND OF THE INVENTION

[0002] The alpha/beta hydrolase fold common to several hydrolyticenzymes of differing phylogenetic origin and catalytic function wasdescribed by Ollis et al. (1992, Protein Eng. 5(3):197-211). The core ofeach enzyme in this family was described as being similar: an alpha/betasheet of eight beta-sheets connected by alpha-helices with conservedarrangement of catalytic residues. Members of this family were found tohave a catalytic triad which is borne on the conserved loop structurefound in the fold. In the five members discussed in Ollis et al., thecatalytic residues always occur in the same order in the primarysequence: nucleophile, acid, histidine. Furthermore, the catalytic triadresidues of the members had similar topological and three dimensionalpositions.

[0003] Members of the hydrolase family include a hydroxylyase (Wajant,et al., 1996, J. Biol.

[0004] Chem. 271(42):25830-25834) which comprises the active site motifGly-X-Ser-X-Gly/Ala and the residues Serine 80, Aspartic 208, andHistidine 236 which are critical for enzyme activity; 2-hydroxymuconicsemialdehyde hydrolase, XylF (Diaz E., 1995, J. Biol. Chem.270(11):6403-6411), which comprises the residues Ser107, Asp228 and His256; non-heme haloperoxidases comprises oxidases, which performhalogenation and which are related to esterases (Pelletier et al., 1995,Biochim Biophys Acta, 1995,1250(2):149-157) which comprises the residuesSerine 97, Aspartic acid 229, Histidine 258; and dipeptidyl-peptidase IV(David et al., 1993, J. Biol. Chem. 268(23): 17247-17252), whichcomprises the residues Ser624Asp702, His734).

SUMMARY OF THE INVENTION

[0005] The present invention relates to the identification ofgram-positive microorganism members of the family of alpha/betahydrolase fold enzymes which are characterized by structural relatednessand which comprises conserved catalytic triads. These newly identifiedmembers of this family can be used in industrial applications, e.g., thetextile industry, in cleaning compositions, such as detergents, and inanimal feeds.

[0006] Accordingly, the present invention provides compositionscomprising a hydrolase selected from the group consisting of YUXL, YTMA,YITV, YQKD, YCLE, YTAP, YDEN, YBFK, YFHM, YDJP, YVFQ, YVAM, YQJL, SRFAD,YCGS, YTPA, YBAC, YUII, YODD, YJCH, YODH which can be used in detergentcompositions, compositions for the treatment of textiles; and animalfeeds, for example. The present invention also provides commercialapplications of the compositions, e.g., their use in methods fortreating textiles and methods for cleaning.

[0007] The present invention provides amino acid sequences forhydrolases obtainable from B. subtilis (B. subtilis hydrolases). Due tothe degeneracy of the genetic code, the present invention encompassesany nucleic acid sequence that encodes the specific hydrolase amino acidsequence shown in the Sequences.

[0008] The present invention provides methods for detecting grampositive microorganism homologs of the B. subtilis hydrolases thatcomprise hybridizing part or all of the nucleic acid encoding thehydrolase with nucleic acid derived from gram-positive organisms, eitherof genomic or cDNA origin. In one embodiment, the gram-positivemicroorganism includes B. licheniformis, B. lentus, B. brevis, B.stearothernophilus, B. alkalophilus, B. amyloliquefaciens, B. coagulans,B. circulans, B. lautus and Bacillus thuringiensis.

[0009] The invention further provides for a hydrolase that has at least80%, at least 85%, at least 90%, and at least 95% homology with thespecific amino acid sequences shown in the sequences. The invention alsoprovides for a nucleic acid which encodes a hydrolase that has at least80%, at least 85%, at least 90% and at least 95% homology with thenaturally occurring nucleic acid sequence found in Bacillus subtilis.

[0010] In a preferred embodiment, the present invention provides thenaturally occurring hydrolase nucleic acid molecule having the sequencefound in Bacillus subtilis 1-168 strain (Bacillus Genetic Stock Center,accession number 1A1, Columbus, Ohio) as disclosed infra.

[0011] The present invention provides expression vectors and host cellscomprising a nucleic acid encoding a gram positive hydrolase. Thepresent invention also provides methods of making the gram positivehydrolase.

[0012] The present invention encompasses novel amino acid variations ofhydrolase amino acid sequences from gram positive microorganismsdisclosed herein that have hydrolytic activity. Naturally occurringhydrolases derived from gram positive microorganisms disclosed herein aswell as proteolytically active amino acid variations or derivativesthereof, have application in the textile industry, in cleaningcompositions and methods and in animal feed compositions.

[0013] In another embodiment, a host cell is engineered to produce ahydrolase of the present invention. In a further aspect of the presentinvention, a hydrolase from a gram positive microorganism is produced onan industrial fermentation scale in a host expression system. The hostcell may be a gram-negative or gram-positive microorganism, a fungalorganism, or higher Eucaryotes. Gram negative microorganisms include butare not limited to members of Enterobacteriaceae; gram positivemicroorganisms include but are not limited to members of Bacillus andPseudomonase; fungal organisms include but are not limited toAspergillus and Tricoderma; and higher eucaryotes include mammaliancells.

[0014] The gram positive microorganism host cell may be normallysporulating or non-sporulating and may be modified in other ways tofacilitate expression of the hydrolase. In a preferred embodiment, thegram positive host cell is a Bacillus. In another embodiment, theBacillus includes B. subtilis, B. licheniformis, B. lentus, B. brevis,B. stearothennophilus, B. alkalophilus, B. amyloliquefaciens, B.coagulans, B. circulans, B. lautus and B. thuringiensis. In a furtherpreferred embodiment, the Bacillus host cell is Bacillus subtilis.

DETAILED DESCRIPITION

[0015] Definitions

[0016] The present invention relates to a newly characterized hydrolasesfrom gram positive organisms. In a preferred embodiment, the grampositive organisms is a Bacillus. In another preferred embodiment, theBacillus includes B. subtilis, B. licheniformis, B. lentus, B. brevis,B. stearothermophilus, B. alkalophilus, B. amyloliquefaciens, B.coagulans, B. circulans, B. lautus and B. thuringiensis.

[0017] In another preferred embodiment, the gram positive organism isBacillus subtilis and the hydrolases have the amino acid sequencedisclosed in the Sequences. In a preferred embodiment, the hydrolase isencoded by the naturally occurring nucleic acid that is found at therespective positions of B. subtilis detailed infra.

[0018] As used herein, “nucleic acid” refers to a nucleotide orpolynucleotide sequence, and fragments or portions thereof, and to DNAor RNA of genomic or synthetic origin which may be double-stranded orsingle-stranded, whether representing the sense or antisense strand. Asused herein “amino acid” refers to peptide or protein sequences orportions thereof. A “polynucleotide homologue” as used herein refers toa gram positive microorganism polynucleotide that has at least 80%, atleast 85%, at least 90% and more preferably at least 95% identity to B.subtilis hydrolase, or which is capable of hybridizing to B. subtilishydrolase under conditions of high stringency and which encodes an aminoacid sequence having hydrolase activity.

[0019] The terms “isolated” or “purified” as used herein refer to anucleic acid or peptide or protein that is removed from at least onecomponent with which it is naturally associated. As used herein,isolated nucleic acid can include a vector comprising the nucleic acid.

[0020] As used herein, the term “overexpressing” when referring to theproduction of a protein in a host cell means that the protein isproduced in greater amounts than in its naturally occurring environment.

[0021] As used herein, the phrase “hydrolytic activity” refers to aprotein that is able to hydrolyze a peptide bond. Enzymes havingproteolytic activity are described in Enzyme Nomenclature, 1992, editedWebb Academic Press, Inc.

[0022] I. Hydrolase Sequences

[0023] The present invention encompasses the use of hydrolasepolynucleotide homologues encoding gram positive microorganismhydrolases which have at least 80%, at least 85%, at least 90%, and atleast 95% identity to naturally occurring B. subtilis hydrolase nucleicacid as long as the homologue encodes a protein that has hydrolyticactivity.

[0024] Gram positive polynucleotide homologues of B. subtilis hydrolasemay be obtained by standard procedures known in the art from, forexample, cloned DNA (e.g., a DNA “library”), genomic DNA libraries, bychemical synthesis once identified, by cDNA cloning, or by the cloningof genomic DNA, or fragments thereof, purified from a desired cell.(See, for example, Sambrook et al., 1989, Molecular Cloning, ALaboratory Manual, 2d Ed., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y.; Glover, D. M. (ed.), 1985, DNA Cloning: A PracticalApproach, MRL Press, Ltd., Oxford, U.K. Vol. I, II.) A preferred sourceis from genomic DNA.

[0025] As will be understood by those of skill in the art, the aminoacid sequence disclosed in the Sequences may reflect inadvertent errorsinherent to nucleic acid sequencing technology. The present inventionencompasses the naturally occurring nucleic acid molecule having thenucleic acid sequence obtained from the genomic sequence of Bacillusspecies and the naturally occurring amino acid sequence.

[0026] Nucleic acid encoding Bacillus subtilis hydrolase starts aroundthe kilobases shown in Table I counting from the point of origin in theBacillus subtilis strain I-168 (Anagnostopala, 1961, J. Bacteriol.,81:741-746 or Bacillus Genomic Stock Center, accession 1A1, Columbus,Ohio). The Bacillus subtilis point of origin has been described inOgasawara, N. (1995, Microbiology 141:Pt.2 257-59). Bacillus subtilishydrolase has a length of 415 amino acids. Based upon the location ofthe DNA encoding Bacillus subtilis hydrolase, naturally occurring B.subtilis hydrolase can be obtained by methods known to those of skill inthe art including PCR technology. The nucleotide sequence for thehydrolases disclosed in Table I can be found in Nature 1997 vol 390,pages 249-256. TABLE I hydrolase designation kb from pt of origin YUXL3111 YTMA 3131 YITV 1190 YQKD 2459 YCLE 414 YTAP 3095 YDEN 573 YDJP 682YVFQ 3496 YQJL 2476 SRFAD 401 YCGS 352 YTPA 3122 YBAC 134 YUII 3291 YODD2128 YODH 2132 YBFK 246 YVAM 3452 YFHM 929

[0027] Oligonucleotide sequences or primers of about 10-30 nucleotidesin length can be designed from the naturally occurring polynucleotidesequences and used in PCR technology to isolate the naturally occurringsequence from B. subtilis genomic sequences.

[0028] Another general strategy for the “cloning” of B. subtilis genomicDNA pieces for sequencing uses inverse PCR. A known region is scannedfor a set of appropriate restriction enzyme cleavage sites and inversePCR is performed with a set of DNA primers determined from the outermostDNA sequence. The DNA fragments from the inverse PCR are directly usedas template in the sequencing reaction. The newly derived sequences canbe used to design new oligonucleotides. These new oligonucleotides areused to amplify DNA fragments with genomic DNA as template. The sequencedetermination on both strands of a DNA region is finished by applying aprimer walking strategy on the genomic PCR fragments. The benefit ofmultiple starting points in the primer walking results from the seriesof inverse PCR fragments with different sizes of new “cloned” DNApieces. From the most external DNA sequence, a new round of inverse PCRis started. The whole inverse PCR strategy is based on the sequentialuse of conventional taq polymerase and the use of long range inverse PCRin those cases in which the taq polymerase failed to amplify DNAfragments. Nucleic acid sequencing is performed using standardtechnology. One method for nucleic acid sequencing involves the use of aPerkin-Elmer Applied Biosystems 373 DNA sequencer (Perkin-Elmer, FosterCity, Calif.) according to manufacturer's instructions.

[0029] Nucleic acid sequences derived from genomic DNA may containregulatory regions in addition to coding regions. Whatever the source,the isolated hydrolase gene should be molecularly cloned into a suitablevector for propagation of the gene.

[0030] In molecular cloning of the gene from genomic DNA, DNA fragmentsare generated, some of which will encode the desired gene. The DNA maybe cleaved at specific sites using various restriction enzymes.Alternatively, one may use DNAse in the presence of manganese tofragment the DNA, or the DNA can be physically sheared, as for example,by sonication. The linear DNA fragments can then be separated accordingto size by standard techniques, including but not limited to, agaroseand polyacrylamide gel electrophoresis and column chromatography.

[0031] Once the DNA fragments are generated, identification of thespecific DNA fragment containing the hydrolase may be accomplished in anumber of ways. For example, a B. subtilis hydrolase gene of the presentinvention or its specific RNA, or a fragment thereof, such as a probe orprimer, may be isolated and labeled and then used in hybridizationassays to detect a gram positive hydrolase gene. (Benton, W. and Davis,R., 1977, Science 196:180; Grunstein, M. and Hogness, D., 1975, Proc.Natl. Acad. Sci. USA 72:3961). Those DNA fragments sharing substantialsequence similarity to the probe will hybridize under stringentconditions.

[0032] Accordingly, the present invention provides a method for thedetection of gram positive hydrolase polynucleotide homologues whichcomprises hybridizing part or all of a nucleic acid sequence of B.subtilis hydrolase with gram positive microorganism nucleic acid ofeither genomic or cDNA origin.

[0033] Also included within the scope of the present invention is theuse of gram positive microorganism polynucleotide sequences that arecapable of hybridizing to the nucleotide sequence of B. subtilishydrolase under conditions of intermediate to maximal stringency.Hybridization conditions are based on the melting temperature (Tm) ofthe nucleic acid binding complex, as taught in Berger and Kimmel (1987,Guide to Molecular Cloning Techniques, Methods in Enzymology, Vol. 152,Academic Press, San Diego, Calif.) incorporated herein by reference, andconfer a defined “stringency” as explained below.

[0034] “Maximum stringency” typically occurs at about Tm−5° C. (5° C.below the Tm of the probe); “high stringency” at about 5° C. to 10° C.below Tm; “intermediate stringency” at about 10° C. to 20° C. below Tm;and “low stringency” at about 20° C. to 25° C. below Tm. As will beunderstood by those of skill in the art, a maximum stringencyhybridization can be used to identify or detect identical polynucleotidesequences while an intermediate or low stringency hybridization can beused to identify or detect polynucleotide sequence homologues.

[0035] The term “hybridization” as used herein shall include “theprocess by which a strand of nucleic acid joins with a complementarystrand through base pairing” (Coombs, J., (1994), Dictionary ofBiotechnology, Stockton Press, New York, N.Y.).

[0036] The process of amplification as carried out in polymerase chainreaction (PCR) technologies is described in Dieffenbach, C W and G SDveksler, (?PCR Primer, a Laboratory Manual, Cold Spring Harbor Press,Plainview, N.Y., 1995). A nucleic acid sequence of at least about 10nucleotides and as many as about 60 nucleotides from B. subtilishydrolase, preferably about 12 to 30 nucleotides, and more preferablyabout 20-25 nucleotides can be used as a probe or PCR primer.

[0037] The B. subtilis hydrolase amino acid sequences shown in theSequences were identified via a BLAST search (Altschul, Stephen, Basiclocal alignment search tool, J. Mol. Biol., 215:403-410) of translatedBacillus subtilis genomic nucleic acid sequences. The conservedcatalytic residues of the hydrolases are illustrated in Table II. TABLEII hydrolase nucleophile acid histidine designation residue residue E/Dresidue YUXL¹ GSY 518 D599 631 YTMA¹ FSR 122 D204 236 YITV¹ TSM 116 D201237 YQKD¹ ESM 160 D249 278 YCLE HSG 95 D232 261 YTAP MSM 169 D244 281YDEN HSL 71 D137 161 YBFK LSL 129 E244 273 YFHM HDW 102 D237 265 YDJPWSM 94 D217 246 YVFQ² HSV 96 D219 247 YVAM² YSA 93 D207 233 YQJL HSY 102SRFAD HSM 86 D 190 H 210 YCGS ISA 68 D 207 H 235 YTPA HSM 88 YBAC HSW115 D 266 H 296 YUII HSL 188 YODD YSN 100 YJCH DSL 129 YODH RSY 172

[0038] II H. Expression Systems

[0039] The present invention provides host cells, expression methods andsystems for the enhanced production and secretion of gram positivemicroorganism hydrolases. In one embodiment of the present invention,the host cell is a gram negative host cell and in another embodiment,the host cell is a gram positive host cell. The host cell may also be afungal or mammalian host cell. In one embodiment of the presentinvention, a gram positive or gram negative microorganism is geneticallyengineered to produce and/or overproduce a hydrolase of the presentinvention.

[0040] III. Production of Hydrolase

[0041] For production of hydrolase in a host cell, an expression vectorcomprising at least one copy of nucleic acid encoding a gram positivemicroorganism hydrolase, and preferably comprising multiple copies, istransformed into the host cell under conditions suitable for expressionof the hydrolase. In accordance with the present invention,polynucleotides which encode a gram positive microorganism hydrolase, orfragments thereof, or fusion proteins or polynucleotide homologuesequences that encode amino acid variants of B. subtilis hydrolase, maybe used to generate recombinant DNA molecules that direct theirexpression in host cells. In a preferred embodiment, the gram positivehost cell belongs to the genus Bacillus. In a further preferredembodiment, the gram positive host cell is B. subtilis.

[0042] As will be understood by those of skill in the art, it may beadvantageous to produce polynucleotide sequences possessingnon-naturally occurring codons. Codons preferred by a particular grampositive host cell (Murray, E. et al., (1989), Nuc. Acids Res.,17:477-508) can be selected, for example, to increase the rate ofexpression or to produce recombinant RNA transcripts having desirableproperties, such as a longer half-life, than transcripts produced from anaturally occurring sequence.

[0043] Altered hydrolase polynucleotide sequences which may be used inaccordance with the invention include deletions, insertions orsubstitutions of different nucleotide residues resulting in apolynucleotide that encodes the same or a functionally equivalenthydrolase homologue, respectively. As used herein a “deletion” isdefined as a change in the nucleotide sequence of the hydrolaseresulting in the absence of one or more amino acid residues.

[0044] As used herein, an “insertion” or “addition” is that change inthe nucleotide sequence which results in the addition of one or moreamino acid residues as compared to the naturally occurring hydrolase.

[0045] As used herein, “substitution” results from the replacement ofone or more nucleotides or amino acids by different nucleotides or aminoacids, respectively. The change(s) in the nucleotides(s) can eitherresult in a change in the amino acid sequence or not.

[0046] The encoded protein may also show deletions, insertions orsubstitutions of amino acid s residues which produce a silent change andresult in a functionally equivalent hydrolase variant. Deliberate aminoacid substitutions may be made on the basis of similarity in polarity,charge, solubility, hydrophobicity, hydrophilicity, and/or theamphipathic nature of the residues as long as the variant retains itsproteolytic ability. For example, negatively charged amino acids includeaspartic acid and glutamic acid; positively charged amino acids includelysine and arginine; and amino acids with uncharged polar head groupshaving similar hydrophilicity values include leucine, isoleucine,valine; glycine, alanine; asparagine, glutamine; serine, threonine,phenylalanine, and tyrosine.

[0047] The hydrolase polynucleotides of the present invention may beengineered in order to modify the cloning, processing and/or expressionof the gene product. For example, mutations may be introduced usingtechniques which are well known in the art, i.e., site-directedmutagenesis to insert new restriction sites, to alter glycosylationpatterns or to change codon preference, for example.

[0048] In one embodiment of the present invention, a gram positivemicroorganism hydrolase polynucleotide may be ligated to a heterologoussequence to encode a fusion protein. A fusion protein may also beengineered to contain a cleavage site located between the hydrolasenucleotide sequence and the heterologous protein sequence, so that thehydrolase may be cleaved and purified away from the heterologous moiety.

[0049] IV. Vector Sequences

[0050] Expression vectors used in expressing the hydrolases of thepresent invention in gram positive microorganisms comprise at least onepromoter associated with the hydrolase, which promoter is functional inthe host cell. In one embodiment of the present invention, the promoteris the wild-type promoter for the selected hydrolase and in anotherembodiment of the present invention, the promoter is heterologous to thehydrolase, but still functional in the host cell. In one preferredembodiment of the present invention, nucleic acid encoding the hydrolaseis stably integrated into the microorganism genome.

[0051] In a preferred embodiment, the expression vector contains amultiple cloning site cassette which preferably comprises at least onerestriction endonuclease site unique to the vector, to facilitate easeof nucleic acid manipulation. In a preferred embodiment, the vector alsocomprises one or more selectable markers. As used herein, the term“selectable marker” refers to a gene capable of expression in the grampositive host which allows for ease of selection of those hostscontaining the vector. Examples of such selectable markers include butare not limited to antibiotics, such as, erythromycin, actinomycin,chloramphenicol and tetracycline.

[0052] V. Transformation

[0053] A variety of host cells can be used for the production Bacillussubtilis hydrolase or hydrolase homologues including bacterial, fungal,mammalian and insects cells. General transformation procedures aretaught in Current Protocols In Molecular Biology, (Vol. 1, edited byAusubel et al., John Wiley & Sons, Inc., 1987, Chapter 9) and includecalcium phosphate methods, transformation using DEAE-Dextran andelectroporation. Plant transformation methods are taught in Rodriquez(WO 95/14099, published May 26, 1995).

[0054] In a preferred embodiment, the host cell is a gram positivemicroorganism and in another preferred embodiment, the host cell isBacillus. In one embodiment of the present invention, nucleic acidencoding one or more hydrolase(s) of the present invention is introducedinto a host cell via an expression vector capable of replicating withinthe Bacillus host cell. Suitable replicating plasmids for Bacillus aredescribed in Molecular Biological Methods for Bacillus, Ed. Harwood andCutting, John Wiley & Sons, 1990, hereby expressly incorporated byreference; see chapter 3 on plasmids. Suitable replicating plasmids forB. subtilis are listed on page 92.

[0055] In another embodiment, nucleic acid encoding a hydrolase(s) ofthe present invention is stably integrated into the microorganismgenome. Preferred host cells are gram positive host cells. Anotherpreferred host is Bacillus. Another preferred host is Bacillus subtilis.Several strategies have been described in the literature for the directcloning of DNA in Bacillus. Plasmid marker rescue transformationinvolves the uptake of a donor plasmid by competent cells carrying apartially homologous resident plasmid (Contente et al., Plasmid,2:555-571 (1979); Haima et al., Mol. Gen. Genet., 223:185-191 (1990);Weinrauch et al., J. Bacteriol., 154(3):1077-1087 (1983); and Weinrauchet al., J. Bacteriol.,169(3):1205-1211 (1987)). The incoming donorplasmid recombines with the homologous region of the resident “helper”plasmid in a process that mimics chromosomal transformation.

[0056] Transformation by protoplast transformation is described for B.subtilis in Chang and Cohen, (1979), Mol. Gen. Genet., 168:111-115; forB. megaterium in Vorobjeva et al., (1980), FEMS Microbiol. Letters,7:261-263; for B. amyloliquefaciens in Smith et al., (1986), Appl. andEnv. Microbiol., 51:634; for B. thuringiensis in Fisher et al., (1981),Arch. Microbiol., 139:213-217; for B. sphaericus in McDonald, (1984), J.Gen. Microbiol., 130:203; and B. larvae in Bakhiet et al., (1985, Appl.Environ. Microbiol. 49:577). Mann et al., (1986, Current Microbiol.,13:131-135) report on transformation of Bacillus protoplasts andHolubova, (1985), Folia Microbiol., 30:97) disclose methods forintroducing DNA into protoplasts using DNA containing liposomes.

[0057] VI. Identification of Transformants

[0058] Whether a host cell has been transformed with a mutated or anaturally occurring gene encoding a gram positive hydrolase detection ofthe presence/absence of marker gene expression can suggest whether thegene of interest is present. However, its expression should beconfirmed. For example, if the nucleic acid encoding a hydrolase isinserted within a marker gene sequence, recombinant cells containing theinsert can be identified by the absence of marker gene function.Alternatively, a marker gene can be placed in tandem with nucleic acidencoding the hydrolase under the control of a single promoter.Expression of the marker gene in response to induction or selectionusually indicates expression of the hydrolase as well.

[0059] Alternatively, host cells which contain the coding sequence for ahydrolase and express the protein may be identified by a variety ofprocedures known to those of skill in the art. These procedures include,but are not limited to, DNA-DNA or DNA-RNA hybridization and proteinbioassay or immunoassay techniques which include membrane-based,solution-based, or chip-based technologies for the detection and/orquantification of the nucleic acid or protein.

[0060] The presence of the hydrolase polynucleotide sequence can bedetected by DNA-DNA or DNA-RNA hybridization or amplification usingprobes, portions or fragments of B. subtilis hydrolase.

[0061] VII. Assay of Activity

[0062] There are various assays known to those of skill in the art fordetecting and measuring hydrolytic activity. There are assays based uponthe release of acid-soluble peptides from casein or hemoglobin measuredas absorbance at 280 nm or calorimetrically using the Folin method(Bergmeyer, et al., 1984, Methods of Enzymatic Analysis, Vol. 5,Peptidases, Proteinases and their Inhibitors, Verlag Chemie, Weinheim).Other assays involve the solubilization of chromogenic substrates (Ward,1983, Proteinases, in Microbial Enzymes and Biotechnology, (W.M.Fogarty, ed.), Applied Science, London, pp. 251-317). Other assays forspecific hydrolases, such as esterases, lipases, peroxidases, are knownby those of skill in the art.

[0063] VIII. Secretion of Recombinant Proteins

[0064] Means for determining the levels of secretion of a heterologousor homologous protein in a gram positive host cell and detectingsecreted proteins include using either polyclonal or monoclonalantibodies specific for the protein. Examples include enzyme-linkedimmunosorbent assay (ELISA), radioimmunoassay (RIA) and fluorescentactivated cell sorting (FACS). These and other assays are described,among other places, in Hampton, R. et al., (1990, Serological Methods, aLaboratory Manual, APS Press, St. Paul Minn.) and Maddox, D E et al.,(1983, J. Exp. Med. 158:1211).

[0065] A wide variety of labels and conjugation techniques are known bythose skilled in the art and can be used in various nucleic and aminoacid assays. Means for producing labeled hybridization or PCR probes fordetecting specific polynucleotide sequences include oligolabeling, nicktranslation, end-labeling or PCR amplification using a labelednucleotide. Alternatively, the nucleotide sequence, or any portion ofit, may be cloned into a vector for the production of an mRNA probe.Such vectors are known in the art, are commercially available, and maybe used to synthesize RNA probes in vitro by addition of an appropriateRNA polymerase such as T7, T3 or SP6 and labeled nucleotides.

[0066] A number of companies such as Pharmacia Biotech (PiscatawayN.J.), Promega (Madison Wis.), and US Biochemical Corp. (Cleveland,Ohio) supply commercial kits and protocols for these procedures.Suitable reporter molecules or labels include those radionuclides,enzymes, fluorescent, chemiluminescent, or chromogenic agents as well assubstrates, cofactors, inhibitors, magnetic particles and the like.Patents teaching the use of such labels include U.S. Pat. Nos.3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149 and4,366,241. Also, recombinant immunoglobulins may be produced as shown inU.S. Pat. No. 4,816,567 and incorporated herein by reference.

[0067] IX. Purification of Proteins

[0068] Gram positive host cells transformed with polynucleotidesequences encoding hydrolases may be cultured under conditions suitablefor the expression and recovery of the hydrolase from cell culture.Other recombinant constructions may join the hydrolase sequences to anucleotide sequence encoding a polypeptide domain which will facilitatepurification of soluble proteins (Kroll, D J. et al., (1993), DNA CellBiol. 12:441-53).

[0069] Such purification facilitating domains include, but are notlimited to, metal chelating peptides such as histidine-tryptophanmodules that allow purification on immobilized metals (Porath, J.,(1992), Protein Expr. Purif. 3:263-281), protein A domains that allowpurification on immobilized immunoglobulin, and the domain utilized inthe FLAGS extension/affinity purification system (Immunex Corp, SeattleWash.). The inclusion of a cleavable linker sequence such as Factor XAor enterokinase (Invitrogen, San Diego, Calif.) between the purificationdomain and the heterologous protein can be used to facilitatepurification.

[0070] X. Uses of The Present Invention

[0071] Hydrolase and Genetically Engineered Host Cells

[0072] The present invention provides genetically engineered host cellscomprising nucleic acid encoding bydrolases of the present invention.The host cell may contain other modifications, e.g., protease deletions,such as deletions of the mature subtilisn protease and/or mature neutralprotease disclosed in U.S. Pat. No. 5,264,366 or other modifications toenhance expression.

[0073] In a preferred embodiment, the host cell is a Bacillus. In afurther preferred embodiment, the host cell is a Bacillus subtilis.

[0074] In a preferred embodiment, the host cell is grown under largescale fermentation conditions. In another preferred embodiment, thehydrolase is isolated and/or purified and used in the textile industry,the feed industry and in cleaning compositions such as detergents.

[0075] As noted, hydrolase can be useful in formulating various cleaningcompositions. A number of known compounds are suitable surfactantsuseful in compositions comprising the hydrolase of the invention. Theseinclude nonionic, anionic, cationic, anionic or zwitterionic detergents,as disclosed in U.S. Pat. No. 4,404,128 and U.S. Pat. No. 4,261,868. Asuitable detergent formulation is that described in Example 7 of U.S.Pat. No. 5,204,015. The art is familiar with the different formulationswhich can be used as cleaning compositions. In addition, hydrolase canbe used, for example, in bar or liquid soap applications, dishcareformulations, contact lens cleaning solutions or products, peptidehydrolysis, waste treatment, textile applications, as fusion-cleavageenzymes in protein production, etc. hydrolase may comprise enhancedperformance in a detergent composition (as compared to another detergentprotease). As used herein, enhanced performance in a detergent isdefined as increasing cleaning of certain enzyme sensitive stains suchas grass or blood, as determined by usual evaluation after a standardwash cycle.

[0076] Hydrolases of the present invention can be formulated into knownpowdered and liquid detergents having pH between 6.5 and 12.0 at levelsof about 0.01 to about 5% (preferably 0.1% to 0.5%) by weight. Thesedetergent cleaning compositions can also include other enzymes such asknown proteases, amylases, cellulases, lipases or endoglycosidases, aswell as builders and stabilizers.

[0077] The addition of hydrolase to conventional cleaning compositionsdoes not create any special use limitation. In other words, anytemperature and pH suitable for the detergent is also suitable for thepresent compositions as long as the pH is within the above range, andthe temperature is below the described hydrolase denaturing temperature.In addition, hydrolase can be used in a cleaning composition withoutdetergents, again either alone or in combination with builders andstabilizers.

[0078] Hydrolases can be included in animal feed such as part of animalfeed additives as described in, for example, U.S. Pat. No. 5,612,055;U.S. Pat. No. 5,314,692; and U.S. Pat. No. 5,147,642.

[0079] One aspect of the invention is a composition for the treatment ofa textile that comprises a hydrolase. The composition can be used totreat for example silk or wool as described in publications such as RD216,034; EP 134,267; U.S. Pat. No. 4,533,359; and EP 344,259.

[0080] Hydrolase Polynucleotides

[0081] A B. subtlis hydrolase polynucleotide, or any part thereof,provides the basis for detecting the presence of gram positivemicroorganism hydrolase polynucleotide homologues through hybridizationtechniques and PCR technology.

[0082] Accordingly, one aspect of the present invention is to providefor nucleic acid hybridization and PCR probes which can be used todetect polynucleotide sequences, including genomic and cDNA sequences,encoding gram positive hydrolase or portions thereof.

[0083] The manner and method of carrying out the present invention maybe more fully is understood by those of skill in the art by reference tothe following examples, which examples are not intended in any manner tolimit the scope of the present invention or of the claims directedthereto

EXAMPLE I Preparation of a Genomic Library

[0084] The following example illustrates the preparation of a Bacillusgenomic library.

[0085] Genomic DNA from Bacillus cells is prepared as taught in CurrentProtocols In Molecular Biology. Vol. 1, edited by Ausubel et al., JohnWiley & Sons, Inc.,1987, Chapter 2. 4.1. Generally, Bacillus cells froma saturated liquid culture are lysed and the proteins removed bydigestion with proteinase K. Cell wall debris, polysaccharides, andremaining proteins are removed by selective precipitation with CTAB, andhigh molecular weight genomic DNA is recovered from the resultingsupernatant by isopropanol precipitation. If exceptionally clean genomicDNA is desired, an additional step of purifying the Bacillus genomic DNAon a cesium chloride gradient is added.

[0086] After obtaining purified genomic DNA, the DNA is subjected toSau3A digestion. Sau3A recognizes the 4 base pair site GATC andgenerates fragments compatible with several convenient phage lambda andcosmid vectors. The DNA is subjected to partial digestion to increasethe chance of obtaining random fragments.

[0087] The partially digested Bacillus genomic DNA is subjected to sizefractionation on a 1% agarose gel prior to cloning into a vector.Alternatively, size fractionation on a sucrose gradient can be used. Thegenomic DNA obtained from the size fractionation step is purified awayfrom the agarose and ligated into a cloning vector appropriate for usein a host cell and transformed into the host cell.

EXAMPLE II Detection of Gram Positive Microorganism Hydrolase

[0088] The following example describes the detection of gram positivemicroorganism hydrolase.

[0089] DNA derived from a gram positive microorganism is preparedaccording to the methods disclosed in Current Protocols in MolecularBiology, Chap. 2 or 3. The nucleic acid is subjected to hybridizationand/or PCR amplification with a probe or primer derived from hydrolase.

[0090] The nucleic acid probe is labeled by combining 50 pmol of thenucleic acid and 250 mCi of [gamma ³²P] adenosine triphosphate(Amersham, Chicago, Ill.) and T4 polynucleotide kinase (DuPont NEN®,Boston Mass.). The labeled probe is purified with Sephadex G-25 superfine resin column (Pharmacia). A portion containing 10⁷ counts perminute of each is used in a typical membrane based hybridizationanalysis of nucleic acid sample of either genomic or cDNA origin.

[0091] The DNA sample which has been subjected to restrictionendonuclease digestion is fractionated on a 0.7 percent agarose gel andtransferred to nylon membranes (Nytran Plus, Schleicher & Schuell,Durham, N.H.). Hybridization is carried out for 16 hours at 40 degreesC. To remove nonspecific signals, blots are sequentially washed at roomtemperature under increasingly stringent conditions up to 0.1× salinesodium citrate and 0.5% sodium dodecyl sulfate. The blots are exposed tofilm for several hours, the film developed and hybridization patternsare compared visually to detect polynucleotide homologues of B. subtilishydrolase. The homologues are subjected to confirmatory nucleic acidsequencing. Methods for nucleic acid sequencing are well known in theart. Conventional enzymatic methods employ DNA polymerase Klenowfragment, SEQUENASE® (US Biochemical Corp, Cleveland, Ohio) or Taqpolymerase to extend DNA chains from an oligonucleotide primer annealedto the DNA template of interest.

[0092] Various other examples and modifications of the foregoingdescription and examples will be apparent to a person skilled in the artafter reading the disclosure without departing from the spirit and scopeof the invention, and it is intended that all such examples ormodifications be included within the scope of the appended claims. Allpublications and patents referenced herein are hereby incorporated byreference in their entirety.

1 21 1 657 PRT Bacillus 1 Met Lys Lys Leu Ile Thr Ala Asp Asp Ile ThrAla Ile Val Ser Val 1 5 10 15 Thr Asp Pro Gln Tyr Ala Pro Asp Gly ThrArg Ala Ala Tyr Val Lys 20 25 30 Ser Gln Val Asn Gln Glu Lys Asp Ser TyrThr Ser Asn Ile Trp Ile 35 40 45 Tyr Glu Thr Lys Thr Gly Gly Ser Val ProTrp Thr His Gly Glu Lys 50 55 60 Arg Ser Thr Asp Pro Arg Trp Ser Pro AspGly Arg Thr Leu Ala Phe 65 70 75 80 Ile Ser Asp Arg Glu Gly Asp Ala AlaGln Leu Tyr Ile Met Ser Thr 85 90 95 Glu Gly Gly Glu Ala Arg Lys Leu ThrAsp Ile Pro Tyr Gly Val Ser 100 105 110 Lys Pro Leu Trp Ser Pro Asp GlyGlu Ser Ile Leu Val Thr Ile Ser 115 120 125 Leu Gly Glu Gly Glu Ser IleAsp Asp Arg Glu Lys Thr Glu Gln Asp 130 135 140 Ser Tyr Glu Pro Val GluVal Gln Gly Leu Ser Tyr Lys Arg Asp Gly 145 150 155 160 Lys Gly Leu ThrArg Gly Ala Tyr Ala Gln Leu Val Leu Val Ser Val 165 170 175 Lys Ser GlyGlu Met Lys Glu Leu Thr Ser His Lys Ala Asp His Gly 180 185 190 Asp ProAla Phe Ser Pro Asp Gly Lys Trp Leu Val Phe Ser Ala Asn 195 200 205 LeuThr Glu Thr Asp Asp Ala Ser Lys Pro His Asp Val Tyr Ile Met 210 215 220Ser Leu Glu Ser Gly Asp Leu Lys Gln Val Thr Pro His Arg Gly Ser 225 230235 240 Phe Gly Ser Ser Ser Phe Ser Pro Asp Gly Arg Tyr Leu Ala Leu Leu245 250 255 Gly Asn Glu Lys Glu Tyr Lys Asn Ala Thr Leu Ser Lys Ala TrpLeu 260 265 270 Tyr Asp Ile Glu Gln Gly Arg Leu Thr Cys Leu Thr Glu MetLeu Asp 275 280 285 Val His Leu Ala Asp Ala Leu Ile Gly Asp Ser Leu IleGly Gly Ala 290 295 300 Glu Gln Arg Pro Ile Trp Thr Lys Asp Ser Gln GlyPhe Tyr Val Ile 305 310 315 320 Gly Thr Asp Gln Gly Ser Thr Gly Ile TyrTyr Ile Ser Ile Glu Gly 325 330 335 Leu Val Tyr Pro Ile Arg Leu Glu LysGlu Tyr Ile Asn Ser Phe Ser 340 345 350 Leu Ser Pro Asp Glu Gln His PheIle Ala Ser Val Thr Lys Pro Asp 355 360 365 Arg Pro Ser Glu Leu Tyr SerIle Pro Leu Gly Gln Glu Glu Lys Gln 370 375 380 Leu Thr Gly Ala Asn AspLys Phe Val Arg Glu His Thr Ile Ser Ile 385 390 395 400 Pro Glu Glu IleGln Tyr Ala Thr Glu Asp Gly Val Met Val Asn Gly 405 410 415 Trp Leu MetArg Pro Ala Gln Met Glu Gly Glu Thr Thr Tyr Pro Leu 420 425 430 Ile LeuAsn Ile His Gly Gly Pro His Met Met Tyr Gly His Thr Tyr 435 440 445 PheHis Glu Phe Gln Val Leu Ala Ala Lys Gly Tyr Ala Val Val Tyr 450 455 460Ile Asn Pro Arg Gly Ser His Gly Tyr Gly Gln Glu Phe Val Asn Ala 465 470475 480 Val Arg Gly Asp Tyr Gly Gly Lys Asp Tyr Asp Asp Val Met Gln Ala485 490 495 Val Asp Glu Ala Ile Lys Arg Asp Pro His Ile Asp Pro Lys ArgLeu 500 505 510 Gly Val Thr Gly Gly Ser Tyr Gly Gly Phe Met Thr Asn TrpIle Val 515 520 525 Gly Gln Thr Asn Arg Phe Lys Ala Ala Val Thr Gln ArgSer Ile Ser 530 535 540 Asn Trp Ile Ser Phe His Gly Val Ser Asp Ile GlyTyr Phe Phe Thr 545 550 555 560 Asp Trp Gln Leu Glu His Asp Met Phe GluAsp Thr Glu Lys Leu Trp 565 570 575 Asp Arg Ser Pro Leu Lys Tyr Ala AlaAsn Val Glu Thr Pro Leu Leu 580 585 590 Ile Leu His Gly Glu Arg Asp AspArg Cys Pro Ile Glu Gln Ala Glu 595 600 605 Gln Leu Phe Ile Ala Leu LysLys Met Gly Lys Glu Thr Lys Leu Val 610 615 620 Arg Phe Pro Asn Ala SerHis Asn Leu Ser Arg Thr Gly His Pro Arg 625 630 635 640 Gln Arg Ile LysArg Leu Asn Tyr Ile Ser Ser Trp Phe Asp Gln His 645 650 655 Leu 2 256PRT Bacillus 2 Met Ile Val Glu Lys Arg Arg Phe Pro Ser Pro Ser Gln HisVal Arg 1 5 10 15 Leu Tyr Thr Ile Cys Tyr Leu Ser Asn Gly Leu Arg ValLys Gly Leu 20 25 30 Leu Ala Glu Pro Ala Glu Pro Gly Gln Tyr Asp Gly PheLeu Tyr Leu 35 40 45 Arg Gly Gly Ile Lys Ser Val Gly Met Val Arg Pro GlyArg Ile Ile 50 55 60 Gln Phe Ala Ser Gln Gly Phe Val Val Phe Ala Pro PheTyr Arg Gly 65 70 75 80 Asn Gln Gly Gly Glu Gly Asn Glu Asp Phe Ala GlyGlu Asp Arg Glu 85 90 95 Asp Ala Phe Ser Ala Phe Arg Leu Leu Gln Gln HisPro Asn Val Lys 100 105 110 Lys Asp Arg Ile His Ile Phe Gly Phe Ser ArgGly Gly Ile Met Gly 115 120 125 Met Leu Thr Ala Ile Glu Met Gly Gly GlnAla Ala Ser Phe Val Ser 130 135 140 Trp Gly Gly Val Ser Asp Met Ile LeuThr Tyr Glu Glu Arg Gln Asp 145 150 155 160 Leu Arg Arg Met Met Lys ArgVal Ile Gly Gly Thr Pro Lys Lys Val 165 170 175 Pro Glu Glu Tyr Gln TrpArg Thr Pro Phe Asp Gln Val Asn Lys Ile 180 185 190 Gln Ala Pro Val LeuLeu Ile His Gly Glu Lys Asp Gln Asn Val Ser 195 200 205 Ile Gln His SerTyr Leu Leu Glu Glu Lys Leu Lys Gln Leu His Lys 210 215 220 Pro Val GluThr Trp Tyr Tyr Ser Thr Phe Thr His Tyr Phe Pro Pro 225 230 235 240 LysGlu Asn Arg Arg Ile Val Arg Gln Leu Thr Gln Trp Met Lys Asn 245 250 2553 255 PRT Bacillus 3 Met Ile Gln Ile Glu Asn Gln Thr Val Ser Gly Ile ProPhe Leu His 1 5 10 15 Ile Val Lys Glu Glu Asn Arg His Arg Ala Val ProLeu Val Ile Phe 20 25 30 Ile His Gly Phe Thr Ser Ala Lys Glu His Asn LeuHis Ile Ala Tyr 35 40 45 Leu Leu Ala Glu Lys Gly Phe Arg Ala Val Leu ProGlu Ala Leu His 50 55 60 His Gly Glu Arg Gly Glu Glu Met Ala Val Glu GluLeu Ala Gly His 65 70 75 80 Phe Trp Asp Ile Val Leu Asn Glu Ile Glu GluIle Gly Val Leu Lys 85 90 95 Asn His Phe Glu Lys Glu Gly Leu Ile Asp GlyGly Arg Ile Gly Leu 100 105 110 Ala Gly Thr Ser Met Gly Gly Ile Thr ThrLeu Gly Ala Leu Thr Ala 115 120 125 Tyr Asp Trp Ile Lys Ala Gly Val SerLeu Met Gly Ser Pro Asn Tyr 130 135 140 Val Glu Leu Phe Gln Gln Gln IleAsp His Ile Gln Ser Gln Gly Ile 145 150 155 160 Glu Ile Asp Val Pro GluGlu Lys Val Gln Gln Leu Met Lys Arg Leu 165 170 175 Glu Leu Arg Asp LeuSer Leu Gln Pro Glu Lys Leu Gln Gln Arg Pro 180 185 190 Leu Leu Phe TrpHis Gly Ala Lys Asp Lys Val Val Pro Tyr Ala Pro 195 200 205 Thr Arg LysPhe Tyr Asp Thr Ile Lys Ser His Tyr Ser Glu Gln Pro 210 215 220 Glu ArgLeu Gln Phe Ile Gly Asp Glu Asn Ala Asp His Lys Val Pro 225 230 235 240Arg Ala Ala Val Leu Lys Thr Ile Glu Trp Phe Glu Thr Tyr Leu 245 250 2554 300 PRT Bacillus 4 Met Lys Lys Ile Leu Leu Ala Ile Gly Ala Leu Val ThrAla Val Ile 1 5 10 15 Ala Ile Gly Ile Val Phe Ser His Met Ile Leu PheIle Lys Lys Lys 20 25 30 Thr Asp Glu Asp Ile Ile Lys Arg Glu Thr Asp AsnGly His Asp Val 35 40 45 Phe Glu Ser Phe Glu Gln Met Glu Lys Thr Ala PheVal Ile Pro Ser 50 55 60 Ala Tyr Gly Tyr Asp Ile Lys Gly Tyr His Val AlaPro His Asp Thr 65 70 75 80 Pro Asn Thr Ile Ile Ile Cys His Gly Val ThrMet Asn Val Leu Asn 85 90 95 Ser Leu Lys Tyr Met His Leu Phe Leu Asp LeuGly Trp Asn Val Leu 100 105 110 Ile Tyr Asp His Arg Arg His Gly Gln SerGly Gly Lys Thr Thr Ser 115 120 125 Tyr Gly Phe Tyr Glu Lys Asp Asp LeuAsn Lys Val Val Ser Leu Leu 130 135 140 Lys Asn Lys Thr Asn His Arg GlyLeu Ile Gly Ile His Gly Glu Ser 145 150 155 160 Met Gly Ala Val Thr AlaLeu Leu Tyr Ala Gly Ala His Cys Ser Asp 165 170 175 Gly Ala Asp Phe TyrIle Ala Asp Cys Pro Phe Ala Cys Phe Asp Glu 180 185 190 Gln Leu Ala TyrArg Leu Arg Ala Glu Tyr Arg Leu Pro Ser Trp Pro 195 200 205 Leu Leu ProIle Ala Asp Phe Phe Leu Lys Leu Arg Gly Gly Tyr Arg 210 215 220 Ala ArgGlu Val Ser Pro Leu Ala Val Ile Asp Lys Ile Glu Lys Pro 225 230 235 240Val Leu Phe Ile His Ser Lys Asp Asp Asp Tyr Ile Pro Val Ser Ser 245 250255 Thr Glu Arg Leu Tyr Glu Lys Lys Arg Gly Pro Lys Ala Leu Tyr Ile 260265 270 Ala Glu Asn Gly Glu His Ala Met Ser Tyr Thr Lys Asn Arg His Thr275 280 285 Tyr Arg Lys Thr Val Gln Glu Phe Leu Asp Asn Met 290 295 3005 281 PRT Bacillus 5 Met Glu Arg Ala Gly Ile Cys His Ser Asp Gly Phe AspLeu Ala Tyr 1 5 10 15 Arg Ile Glu Gly Glu Gly Ala Pro Ile Leu Val IleGly Ser Ala Ala 20 25 30 Tyr Tyr Pro Arg Leu Phe Ser Ser Asp Ile Lys GlnLys Tyr Gln Trp 35 40 45 Val Phe Val Asp His Arg Gly Phe Ala Lys Pro LysArg Glu Leu Arg 50 55 60 Ala Glu Asp Ser Arg Leu Asp Ala Val Leu Ala AspIle Glu Arg Met 65 70 75 80 Arg Thr Phe Leu Gln Leu Glu Asp Val Thr ThrLeu Gly His Ser Gly 85 90 95 His Ala Phe Met Ala Leu Glu Tyr Ala Arg ThrTyr Pro Lys Gln Val 100 105 110 Arg Lys Val Ala Leu Phe Asn Thr Ala ProAsp Asn Ser Glu Glu Arg 115 120 125 Gln Arg Lys Ser Glu Ser Phe Phe MetGlu Thr Ala Ser Leu Glu Arg 130 135 140 Lys Lys Arg Phe Glu Lys Asp IleGlu Asn Leu Pro Gln Asp Ile Asp 145 150 155 160 Lys Asp Pro Glu Arg ArgPhe Val His Met Cys Ile Arg Ala Glu Ala 165 170 175 Lys Ser Phe Tyr GlnGlu Arg Pro His Ala Ala Ala Leu Trp Asp Gly 180 185 190 Val Phe Thr AsnMet Pro Ile Ile Asp Glu Leu Trp Gly Asn Thr Phe 195 200 205 Ala Arg IleAsp Leu Leu Gln Arg Leu Ala Asp Val Arg Met Pro Val 210 215 220 Tyr IleGly Leu Gly Arg Tyr Asp Tyr Leu Val Ala Pro Val Ser Leu 225 230 235 240Trp Asp Ala Val Asp Gly Leu Tyr Pro His Val Asp Lys Val Ile Phe 245 250255 Glu Lys Ser Gly His Gln Pro Met Leu Glu Glu Pro Glu Ala Phe Asp 260265 270 Gln Ser Phe Arg Lys Trp Leu Asp Gln 275 280 6 298 PRT Bacillus 6Met Arg Ala Glu Arg Arg Lys Gln Leu Phe Arg Leu Leu Gly Asp Leu 1 5 1015 Pro Asp Arg Arg Pro Ile Ser Val Glu Thr Leu Arg Ile Glu Glu Arg 20 2530 Glu Glu Asn Ile Val Glu Thr Leu Leu Leu Asp Leu Asn Gly His Glu 35 4045 Lys Ala Pro Ala Tyr Phe Val Lys Pro Lys Lys Thr Glu Gly Pro Cys 50 5560 Pro Ala Val Leu Phe Gln His Ser His Gly Gly Gln Tyr Asp Arg Gly 65 7075 80 Lys Ser Glu Leu Ile Glu Gly Ala Asp Tyr Leu Lys Thr Pro Ser Phe 8590 95 Ser Asp Glu Leu Thr Ser Leu Gly Tyr Gly Val Leu Ala Ile Asp His100 105 110 Trp Gly Phe Gly Asp Arg Arg Gly Lys Ala Glu Ser Glu Ile PheLys 115 120 125 Glu Met Leu Leu Thr Gly Lys Val Met Trp Gly Met Met IleTyr Asp 130 135 140 Ser Leu Ser Ala Leu Asp Tyr Met Gln Ser Arg Ser AspVal Gln Pro 145 150 155 160 Asp Arg Ile Gly Thr Ile Gly Met Ser Met GlyGly Leu Met Ala Trp 165 170 175 Trp Thr Ala Ala Leu Asp Asp Arg Ile LysVal Cys Val Asp Leu Cys 180 185 190 Ser Gln Val Asp His His Val Leu IleLys Thr Gln Asn Leu Asp Arg 195 200 205 His Gly Phe Tyr Tyr Tyr Val ProSer Leu Ala Lys His Phe Ser Ala 210 215 220 Ser Glu Ile Gln Ser Leu IleAla Pro Arg Pro His Leu Ser Leu Val 225 230 235 240 Gly Val His Asp ArgLeu Thr Pro Ala Glu Gly Val Asp Lys Ile Glu 245 250 255 Lys Glu Leu ThrAla Val Tyr Ala Gly Gln Gly Ala Ala Asp Cys Tyr 260 265 270 Arg Val ValArg Ser Ala Ser Gly His Phe Glu Thr Ala Val Ile Arg 275 280 285 His GluAla Val Arg Phe Leu Gln Lys Trp 290 295 7 190 PRT Bacillus 7 Met Thr LysGln Val Tyr Ile Ile His Gly Tyr Arg Ala Ser Ser Thr 1 5 10 15 Asn HisTrp Phe Pro Trp Leu Lys Lys Arg Leu Leu Ala Asp Gly Val 20 25 30 Gln AlaAsp Ile Leu Asn Met Pro Asn Pro Leu Gln Pro Arg Leu Glu 35 40 45 Asp TrpLeu Asp Thr Leu Ser Leu Tyr Gln His Thr Leu His Glu Asn 50 55 60 Thr TyrLeu Val Ala His Ser Leu Gly Cys Pro Ala Ile Leu Arg Phe 65 70 75 80 LeuGlu His Leu Gln Leu Arg Lys Gln Leu Gly Gly Ile Ile Leu Val 85 90 95 SerGly Phe Ala Lys Ser Leu Pro Thr Leu Gln Met Leu Asp Glu Phe 100 105 110Thr Gln Gly Ser Phe Asp His Gln Lys Ile Ile Glu Ser Ala Lys His 115 120125 Arg Ala Val Ile Ala Ser Lys Asp Asp Gln Ile Val Pro Phe Ser Phe 130135 140 Ser Lys Asp Leu Ala Gln Gln Ile Asp Ala Ala Leu Tyr Glu Val Gln145 150 155 160 His Gly Gly His Phe Leu Glu Asp Glu Gly Phe Thr Ser LeuPro Ile 165 170 175 Val Tyr Asp Val Leu Thr Ser Tyr Phe Ser Lys Glu ThrArg 180 185 190 8 296 PRT Bacillus 8 Met Ile Gln Asp Ser Met Gln Phe AlaAla Val Glu Ser Gly Leu Arg 1 5 10 15 Phe Tyr Gln Ala Tyr Asp Gln SerLeu Ser Leu Trp Pro Ile Glu Ser 20 25 30 Glu Ala Phe Tyr Val Ser Thr ArgPhe Gly Lys Thr His Ile Ile Ala 35 40 45 Ser Gly Pro Lys Asp Ala Pro SerLeu Ile Leu Leu His Gly Gly Leu 50 55 60 Phe Ser Ser Ala Met Trp Tyr ProAsn Ile Ala Ala Trp Ser Ser Gln 65 70 75 80 Phe Arg Thr Tyr Ala Val AspIle Ile Gly Asp Lys Asn Lys Ser Ile 85 90 95 Pro Ser Ala Ala Met Glu ThrArg Ala Asp Phe Ala Glu Trp Met Lys 100 105 110 Asp Val Phe Asp Ser LeuGly Leu Glu Thr Ala His Leu Ala Gly Leu 115 120 125 Ser Leu Gly Gly SerHis Ile Val Asn Phe Leu Leu Arg Ala Pro Glu 130 135 140 Arg Val Glu ArgAla Val Val Ile Ser Pro Ala Glu Ala Phe Ile Ser 145 150 155 160 Phe HisPro Asp Val Tyr Lys Tyr Ala Ala Glu Leu Thr Gly Ala Arg 165 170 175 GlyAla Glu Ser Tyr Ile Lys Trp Ile Thr Gly Asp Ser Tyr Asp Leu 180 185 190His Pro Leu Leu Gln Arg Gln Ile Val Ala Gly Val Glu Trp Gln Asp 195 200205 Glu Gln Arg Ser Leu Lys Pro Thr Glu Asn Gly Phe Pro Tyr Val Phe 210215 220 Thr Asp Gln Glu Leu Lys Ser Ile Gln Val Pro Val Leu Leu Met Phe225 230 235 240 Gly Glu His Glu Ala Met Tyr His Gln Gln Met Ala Phe GluArg Ala 245 250 255 Ser Val Leu Val Pro Gly Ile Gln Ala Glu Ile Val LysAsn Ala Gly 260 265 270 His Leu Leu Ser Leu Glu Gln Pro Glu Tyr Val AsnGln Arg Val Leu 275 280 285 Ser Phe Leu Cys Gly Gly Ile Lys 290 295 9286 PRT Bacillus 9 Met Asp Gly Val Lys Cys Gln Phe Val Asn Thr Asn GlyIle Thr Leu 1 5 10 15 His Val Ala Ala Ala Gly Arg Glu Asp Gly Pro LeuIle Val Leu Leu 20 25 30 His Gly Phe Pro Glu Phe Trp Tyr Gly Trp Lys AsnGln Ile Lys Pro 35 40 45 Leu Val Asp Ala Gly Tyr Arg Val Ile Ala Pro AspGln Arg Gly Tyr 50 55 60 Asn Leu Ser Asp Lys Pro Glu Gly Ile Asp Ser TyrArg Ile Asp Thr 65 70 75 80 Leu Arg Asp Asp Ile Ile Gly Leu Ile Thr GlnPhe Thr Asp Glu Lys 85 90 95 Ala Ile Val Ile Gly His Asp Trp Gly Gly AlaVal Ala Trp His Leu 100 105 110 Ala Ser Thr Arg Pro Glu Tyr Leu Glu LysLeu Ile Ala Ile Asn Ile 115 120 125 Pro His Pro His Val Met Lys Thr ValThr Pro Leu Tyr Pro Pro Gln 130 135 140 Trp Leu Lys Ser Ser Tyr Ile AlaTyr Phe Gln Leu Pro Asp Ile Pro 145 150 155 160 Glu Ala Ser Leu Arg GluAsn Asp Tyr Asp Thr Leu Asp Lys Ala Ile 165 170 175 Gly Leu Ser Asp ArgPro Ala Leu Phe Thr Ser Glu Asp Val Ser Arg 180 185 190 Tyr Lys Glu AlaTrp Lys Gln Pro Gly Ala Leu Thr Ala Met Leu Asn 195 200 205 Trp Tyr ArgAla Leu Arg Lys Gly Ser Leu Ala Glu Lys Pro Ser Tyr 210 215 220 Glu ThrVal Pro Tyr Arg Met Ile Trp Gly Met Glu Asp Arg Phe Leu 225 230 235 240Ser Arg Lys Leu Ala Lys Glu Thr Glu Arg His Cys Pro Asn Gly His 245 250255 Leu Ile Phe Val Asp Glu Ala Ser His Trp Ile Asn His Glu Lys Pro 260265 270 Ala Ile Val Asn Gln Leu Ile Leu Glu Tyr Leu Lys Asn Gln 275 280285 10 271 PRT Bacillus 10 Met Pro Tyr Ile Ile Leu Glu Asp Gln Thr ArgLeu Tyr Tyr Glu Thr 1 5 10 15 His Gly Ser Gly Thr Pro Ile Leu Phe IleHis Gly Val Leu Met Ser 20 25 30 Gly Gln Phe Phe His Lys Gln Phe Ser ValLeu Ser Ala Asn Tyr Gln 35 40 45 Cys Ile Arg Leu Asp Leu Arg Gly His GlyGlu Ser Asp Lys Val Leu 50 55 60 His Gly His Thr Ile Ser Gln Tyr Ala ArgAsp Ile Arg Glu Phe Leu 65 70 75 80 Asn Ala Met Glu Leu Asp His Val ValLeu Ala Gly Trp Ser Met Gly 85 90 95 Ala Phe Val Val Trp Asp Tyr Leu AsnGln Phe Gly Asn Asp Asn Ile 100 105 110 Gln Ala Ala Val Ile Ile Asp GlnSer Ala Ser Asp Tyr Gln Trp Glu 115 120 125 Gly Trp Glu His Gly Pro PheAsp Phe Asp Gly Leu Lys Thr Ala Met 130 135 140 His Ala Ile Gln Thr AspPro Leu Pro Phe Tyr Glu Ser Phe Ile Gln 145 150 155 160 Asn Met Phe AlaGlu Pro Pro Ala Glu Thr Glu Thr Glu Trp Met Leu 165 170 175 Ala Glu IleLeu Lys Gln Pro Ala Ala Ile Ser Ser Thr Ile Leu Phe 180 185 190 Asn GlnThr Ala Ala Asp Tyr Arg Gly Thr Leu Gln Asn Ile Asn Val 195 200 205 ProAla Leu Leu Cys Phe Gly Glu Asp Lys Lys Phe Phe Ser Thr Ala 210 215 220Ala Gly Glu His Leu Arg Ser Asn Ile Pro Asn Ala Thr Leu Val Thr 225 230235 240 Phe Pro Lys Ser Ser His Cys Pro Phe Leu Glu Glu Pro Asp Ala Phe245 250 255 Asn Ser Thr Leu Leu Ser Phe Leu Asp Gly Val Ile Gly Lys Ser260 265 270 11 269 PRT Bacillus 11 Met Asn Glu Ala Ile Leu Ser Arg AsnHis Val Lys Val Lys Gly Ser 1 5 10 15 Gly Lys Ala Ser Ile Met Phe AlaPro Gly Phe Gly Cys Asp Gln Ser 20 25 30 Val Trp Asn Ala Val Ala Pro AlaPhe Glu Glu Asp His Arg Val Ile 35 40 45 Leu Phe Asp Tyr Val Gly Ser GlyHis Ser Asp Leu Arg Ala Tyr Asp 50 55 60 Leu Asn Arg Tyr Gln Thr Leu AspGly Tyr Ala Gln Asp Val Leu Asp 65 70 75 80 Val Cys Glu Ala Leu Asp LeuLys Glu Thr Val Phe Val Gly His Ser 85 90 95 Val Gly Ala Leu Ile Gly MetLeu Ala Ser Ile Arg Arg Pro Glu Leu 100 105 110 Phe Ser His Leu Val MetVal Gly Pro Ser Pro Cys Tyr Leu Asn Asp 115 120 125 Pro Pro Glu Tyr TyrGly Gly Phe Glu Glu Glu Gln Leu Leu Gly Leu 130 135 140 Leu Glu Met MetGlu Lys Asn Tyr Ile Gly Trp Ala Thr Val Phe Ala 145 150 155 160 Ala ThrVal Leu Asn Gln Pro Asp Arg Pro Glu Ile Lys Glu Glu Leu 165 170 175 GluSer Arg Phe Cys Ser Thr Asp Pro Val Ile Ala Arg Gln Phe Ala 180 185 190Lys Ala Ala Phe Phe Ser Asp His Arg Glu Asp Leu Ser Lys Val Thr 195 200205 Val Pro Ser Leu Ile Leu Gln Cys Ala Asp Asp Ile Ile Ala Pro Ala 210215 220 Thr Val Gly Lys Tyr Met His Gln His Leu Pro Tyr Ser Ser Leu Lys225 230 235 240 Gln Met Glu Ala Arg Gly His Cys Pro His Met Ser His ProAsp Glu 245 250 255 Thr Ile Gln Leu Ile Gly Asp Tyr Leu Lys Ala His Val260 265 12 256 PRT Bacillus 12 Met Pro Leu Ile Ser Ile Asp Ser Arg LysHis Leu Phe Tyr Glu Glu 1 5 10 15 Tyr Gly Gln Gly Ile Pro Ile Ile PheIle His Pro Pro Gly Met Gly 20 25 30 Arg Lys Val Phe Tyr Tyr Gln Arg LeuLeu Ser Lys His Phe Arg Val 35 40 45 Ile Phe Pro Asp Leu Ser Gly His GlyAsp Ser Asp His Ile Asp Gln 50 55 60 Pro Ala Ser Ile Ser Tyr Tyr Ala AsnGlu Ile Ala Gln Phe Met Asp 65 70 75 80 Ala Leu His Ile Asp Lys Ala ValLeu Phe Gly Tyr Ser Ala Gly Gly 85 90 95 Leu Ile Ala Gln His Ile Gly PheThr Arg Pro Asp Lys Val Ser His 100 105 110 Leu Ile Leu Ser Gly Ala TyrPro Ala Val His Asn Val Ile Gly Gln 115 120 125 Lys Leu His Lys Leu GlyMet Tyr Leu Leu Glu Lys Asn Pro Gly Leu 130 135 140 Leu Met Lys Ile LeuAla Gly Ser His Thr Lys Asp Arg Gln Leu Arg 145 150 155 160 Ser Ile LeuThr Asp His Met Lys Lys Ala Asp Gln Ala His Trp His 165 170 175 Gln TyrTyr Leu Asp Ser Leu Gly Tyr Asn Cys Ile Glu Gln Leu Pro 180 185 190 ArgLeu Glu Met Pro Met Leu Phe Met Tyr Gly Gly Leu Arg Asp Trp 195 200 205Thr Phe Thr Asn Ala Gly Tyr Tyr Arg Arg Ser Cys Arg His Ala Glu 210 215220 Phe Phe Arg Leu Glu Tyr Gln Gly His Gln Leu Pro Thr Lys Gln Trp 225230 235 240 Lys Thr Cys Asn Glu Leu Val Thr Gly Phe Val Leu Thr His HisSer 245 250 255 13 253 PRT Bacillus 13 Met Lys Ser Ala Trp Met Glu LysThr Tyr Thr Ile Asp Gly Cys Ala 1 5 10 15 Phe His Thr Gln His Arg LysGly Ser Ser Gly Val Thr Ile Val Phe 20 25 30 Glu Ala Gly Tyr Gly Thr SerSer Glu Thr Trp Lys Pro Leu Met Ala 35 40 45 Asp Ile Asp Asp Glu Phe GlyIle Phe Thr Tyr Asp Arg Ala Gly Ile 50 55 60 Gly Lys Ser Gly Gln Ser ArgAla Lys Arg Thr Ala Asp Gln Gln Val 65 70 75 80 Lys Glu Leu Glu Ser LeuLeu Lys Ala Ala Asp Val Lys Pro Pro Tyr 85 90 95 Leu Ala Val Ser His SerTyr Gly Ala Val Ile Thr Gly Leu Trp Ala 100 105 110 Cys Lys Asn Lys HisAsp Ile Ile Gly Met Val Leu Leu Asp Pro Ala 115 120 125 Leu Gly Asp CysAla Ser Phe Thr Phe Ile Pro Glu Glu Met His Lys 130 135 140 Ser His ThrArg Lys Met Met Leu Glu Gly Thr His Ala Glu Phe Ser 145 150 155 160 LysSer Leu Gln Glu Leu Lys Lys Arg Gln Val His Leu Gly Asn Met 165 170 175Pro Leu Leu Val Leu Ser Ser Gly Glu Arg Thr Glu Lys Phe Ala Ala 180 185190 Glu Gln Glu Trp Gln Asn Leu His Ser Ser Ile Leu Ser Leu Ser Asn 195200 205 Gln Ser Gly Trp Ile Gln Ala Lys Asn Ser Ser His Asn Ile His His210 215 220 Asp Glu Pro His Ile Val His Leu Ala Ile Tyr Asp Val Trp CysAla 225 230 235 240 Ala Cys Gln Gln Ala Ala Pro Leu Tyr Gln Ala Val Asn245 250 14 242 PRT Bacillus 14 Met Ser Gln Leu Phe Lys Ser Phe Asp AlaSer Glu Lys Thr Gln Leu 1 5 10 15 Ile Cys Phe Pro Phe Ala Gly Gly TyrSer Ala Ser Phe Arg Pro Leu 20 25 30 His Ala Phe Leu Gln Gly Glu Cys GluMet Leu Ala Ala Glu Pro Pro 35 40 45 Gly His Gly Thr Asn Gln Thr Ser AlaIle Glu Asp Leu Glu Glu Leu 50 55 60 Thr Asp Leu Tyr Lys Gln Glu Leu AsnLeu Arg Pro Asp Arg Pro Phe 65 70 75 80 Val Leu Phe Gly His Ser Met GlyGly Met Ile Thr Phe Arg Leu Ala 85 90 95 Gln Lys Leu Glu Arg Glu Gly IlePhe Pro Gln Ala Val Ile Ile Ser 100 105 110 Ala Ile Gln Pro Pro His IleGln Arg Lys Lys Val Ser His Leu Pro 115 120 125 Asp Asp Gln Phe Leu AspHis Ile Ile Gln Leu Gly Gly Met Pro Ala 130 135 140 Glu Leu Val Glu AsnLys Glu Val Met Ser Phe Phe Leu Pro Ser Phe 145 150 155 160 Arg Ser AspTyr Arg Ala Leu Glu Gln Phe Glu Leu Tyr Asp Leu Ala 165 170 175 Gln IleGln Ser Pro Val His Val Phe Asn Gly Leu Asp Asp Lys Lys 180 185 190 CysIle Arg Asp Ala Glu Gly Trp Lys Lys Trp Ala Lys Asp Ile Thr 195 200 205Phe His Gln Phe Asp Gly Gly His Met Phe Leu Leu Ser Gln Thr Glu 210 215220 Glu Val Ala Glu Arg Ile Phe Ala Ile Leu Asn Gln His Pro Ile Ile 225230 235 240 Gln Pro 15 256 PRT Bacillus 15 Met His Gly Gly His Ser AsnCys Tyr Glu Glu Phe Gly Tyr Thr Ala 1 5 10 15 Leu Ile Glu Gln Gly TyrSer Ile Ile Thr Pro Ser Arg Pro Gly Tyr 20 25 30 Gly Arg Thr Ser Lys GluIle Gly Lys Ser Leu Ala Asn Ala Cys Arg 35 40 45 Phe Tyr Val Lys Leu LeuAsp His Leu Gln Ile Glu Ser Val His Val 50 55 60 Ile Ala Ile Ser Ala GlyGly Pro Ser Gly Ile Cys Phe Ala Ser His 65 70 75 80 Tyr Pro Glu Arg ValAsn Thr Leu Thr Leu Gln Ser Ala Val Thr Lys 85 90 95 Glu Trp Leu Thr ProLys Asp Thr Glu Tyr Lys Leu Gly Glu Ile Leu 100 105 110 Phe Arg Pro ProVal Glu Lys Trp Ile Trp Lys Leu Ile Ser Ser Leu 115 120 125 Asn Asn AlaPhe Pro Arg Leu Met Phe Arg Ala Met Ser Pro Gln Phe 130 135 140 Ser ThrLeu Pro Phe Gln Arg Ile Lys Ser Leu Met Asn Glu Lys Asp 145 150 155 160Ile Glu Ala Phe Arg Lys Met Asn Ser Arg Gln Arg Ser Gly Glu Gly 165 170175 Phe Leu Ile Asp Leu Ser Gln Thr Ala Ala Val Ser Leu Lys Asp Leu 180185 190 Gln Ala Ile Ile Cys Pro Val Leu Ile Met Gln Ser Val Tyr Asp Gly195 200 205 Leu Val Asp Leu Ser His Ala His His Ala Lys Glu His Ile ArgGly 210 215 220 Ala Val Leu Cys Leu Leu His Ser Trp Gly His Leu Ile TrpLeu Gly 225 230 235 240 Lys Glu Ala Ala Glu Thr Gly Ser Ile Leu Leu GlyPhe Leu Glu Ser 245 250 255 16 318 PRT Bacillus 16 Met Ile Pro Glu LysLys Ser Ile Ala Ile Met Lys Glu Leu Ser Ile 1 5 10 15 Gly Asn Thr LysGln Met Leu Met Ile Asn Gly Val Asp Val Lys Asn 20 25 30 Pro Leu Leu LeuPhe Leu His Gly Gly Pro Gly Thr Pro Gln Ile Gly 35 40 45 Tyr Val Arg HisTyr Gln Lys Glu Leu Glu Gln Tyr Phe Thr Val Val 50 55 60 His Trp Asp GlnArg Gly Ser Gly Leu Ser Tyr Ser Lys Arg Ile Ser 65 70 75 80 His His SerMet Thr Ile Asn His Phe Ile Lys Asp Thr Ile Gln Val 85 90 95 Thr Gln TrpLeu Leu Ala His Phe Ser Lys Ser Lys Leu Tyr Leu Ala 100 105 110 Gly HisSer Trp Gly Ser Ile Leu Ala Leu His Val Leu Gln Gln Arg 115 120 125 ProAsp Leu Phe Tyr Thr Tyr Tyr Gly Ile Ser Gln Val Val Asn Pro 130 135 140Gln Asp Glu Glu Ser Thr Ala Tyr Gln His Ile Arg Glu Ile Ser Glu 145 150155 160 Ser Lys Lys Ala Ser Ile Leu Ser Phe Leu Thr Arg Phe Ile Gly Ala165 170 175 Pro Pro Trp Lys Gln Asp Ile Gln His Leu Ile Tyr Arg Phe CysVal 180 185 190 Glu Leu Thr Arg Gly Gly Phe Thr His Arg His Arg Gln SerLeu Ala 195 200 205 Val Leu Phe Gln Met Leu Thr Gly Asn Glu Tyr Gly ValArg Asn Met 210 215 220 His Ser Phe Leu Asn Gly Leu Arg Phe Ser Lys LysHis Leu Thr Asp 225 230 235 240 Glu Leu Tyr Arg Phe Asn Ala Phe Thr SerVal Pro Ser Ile Lys Val 245 250 255 Pro Cys Val Phe Ile Ser Gly Lys HisAsp Leu Ile Val Pro Ala Glu 260 265 270 Ile Ser Lys Gln Tyr Tyr Gln GluLeu Glu Ala Pro Glu Lys Arg Trp 275 280 285 Phe Gln Phe Glu Asn Ser AlaHis Thr Pro His Ile Glu Glu Pro Ser 290 295 300 Leu Phe Ala Asn Thr LeuSer Arg His Ala Arg Asn His Leu 305 310 315 17 314 PRT Bacillus 17 MetArg Cys Leu Val Asp Ser Glu Asn His Tyr His Thr Leu Arg Phe 1 5 10 15Ser Leu Arg Arg Gly Met Ser Tyr Cys Met Lys Glu Gln Thr Thr Asp 20 25 30Arg Thr Asn Gly Gly Thr Ser Asn Ala Phe Thr Ile Pro Gly Thr Glu 35 40 45Val Arg Met Met Ser Ser Arg Asn Glu Asn Arg Thr Tyr His Ile Phe 50 55 60Ile Ser Lys Pro Ser Thr Pro Pro Pro Pro Ala Gly Tyr Pro Val Ile 65 70 7580 Tyr Leu Leu Asp Ala Asn Ser Val Phe Gly Thr Met Thr Glu Ala Val 85 9095 Arg Ile Gln Gly Arg Arg Pro Glu Lys Thr Gly Val Ile Pro Ala Val 100105 110 Ile Val Gly Ile Gly Tyr Glu Thr Ala Glu Pro Phe Ser Ser Ala Arg115 120 125 His Arg Asp Phe Thr Met Pro Thr Ala Gln Ser Lys Leu Pro GluArg 130 135 140 Pro Asp Gly Arg Glu Trp Pro Glu His Gly Gly Ala Glu GlyPhe Phe 145 150 155 160 Arg Phe Ile Glu Glu Asp Leu Lys Pro Glu Ile GluArg Asp Tyr Gln 165 170 175 Ile Asp Lys Lys Arg Gln Thr Ile Phe Gly HisSer Leu Gly Gly Leu 180 185 190 Phe Val Leu Gln Val Leu Leu Thr Lys ProAsp Ala Phe Gln Thr Tyr 195 200 205 Ile Ala Gly Ser Pro Ser Ile His TrpAsn Lys Pro Phe Ile Leu Lys 210 215 220 Lys Thr Asp His Phe Val Ser LeuThr Lys Lys Asn Asn Gln Pro Ile 225 230 235 240 Asn Ile Leu Leu Ala AlaGly Glu Leu Glu Gln His His Lys Ser Arg 245 250 255 Met Asn Asp Asn AlaArg Glu Leu Tyr Glu Arg Leu Ala Val Leu Ser 260 265 270 Glu Gln Gly IleArg Ala Glu Phe Cys Glu Phe Ser Gly Glu Gly His 275 280 285 Ile Ser ValLeu Pro Val Leu Val Ser Arg Ala Leu Arg Phe Ala Leu 290 295 300 His ProAsp Gly Pro His Leu Ser Met Gly 305 310 18 200 PRT Bacillus 18 Met LysHis Ile Tyr Glu Lys Gly Thr Ser Asp Asn Val Leu Leu Leu 1 5 10 15 LeuHis Gly Thr Gly Gly Asn Glu His Asp Leu Leu Ser Leu Gly Arg 20 25 30 PheIle Asp Pro Asp Ala His Leu Leu Gly Val Arg Gly Ser Val Leu 35 40 45 GluAsn Gly Met Pro Arg Phe Phe Lys Arg Leu Ser Glu Gly Val Phe 50 55 60 AspGlu Lys Asp Leu Val Val Arg Thr Arg Glu Leu Lys Asp Phe Ile 65 70 75 80Asp Glu Ala Ala Glu Thr His Gln Phe Asn Arg Gly Arg Val Ile Ala 85 90 95Val Gly Tyr Ser Asn Gly Ala Asn Ile Ala Ala Ser Leu Leu Phe His 100 105110 Tyr Lys Asp Val Leu Lys Gly Ala Ile Leu His His Pro Met Val Pro 115120 125 Ile Arg Gly Ile Glu Leu Pro Asp Met Ala Gly Leu Pro Val Phe Ile130 135 140 Gly Ala Gly Lys Tyr Asp Pro Leu Cys Thr Lys Glu Glu Ser GluGlu 145 150 155 160 Leu Tyr Arg Tyr Leu Arg Asp Ser Gly Ala Ser Ala SerVal Tyr Trp 165 170 175 Gln Asp Gly Gly His Gln Leu Thr Gln His Glu AlaGlu Gln Ala Arg 180 185 190 Glu Trp Tyr Lys Glu Ala Ile Val 195 200 19240 PRT Bacillus 19 Met Ala Pro Lys Asn Gly Thr Val Gln Glu Lys Lys PhePhe Ser Lys 1 5 10 15 Glu Leu Asn Glu Glu Met Thr Leu Leu Val Tyr LeuPro Ser Asn Tyr 20 25 30 Ser Pro Leu Tyr Lys Tyr His Val Ile Ile Ala GlnAsp Gly His Asp 35 40 45 Tyr Phe Arg Leu Gly Arg Ile Gly Arg Gln Val GluGlu Leu Leu Ser 50 55 60 Lys Arg Glu Ile Asp Arg Ser Ile Ile Ile Gly ValPro Tyr Lys Asp 65 70 75 80 Val Lys Glu Arg Arg Asn Thr Tyr His Pro GluGly Ser Lys Phe Ser 85 90 95 Ala Tyr Lys Arg Phe Ile Ala His Glu Leu ValPro Phe Ala Asp Asp 100 105 110 Glu Tyr Pro Thr Tyr Gln Ile Gly Tyr GlyArg Thr Leu Ile Gly Asp 115 120 125 Ser Leu Gly Ala Thr Val Ser Leu MetThr Ala Leu Asp Tyr Pro Asn 130 135 140 Met Phe Gly Asn Ile Ile Met GlnSer Pro Tyr Val Asp Lys His Val 145 150 155 160 Leu Glu Ala Val Lys GlnSer Asp Asp Ile Lys His Leu Ser Ile Tyr 165 170 175 His Gln Ile Gly ThrLys Glu Thr Asp Val His Thr Thr Asp Gly Asn 180 185 190 Ile Leu Asp PheThr Glu Pro Asn Arg Glu Leu Lys Gln Leu Leu Glu 195 200 205 Lys Lys LeuSer Asp Tyr Asp Phe Glu Pro Phe Asp Gly Asp His Lys 210 215 220 Trp ThrTyr Trp Gln Pro Leu Ile Thr Pro Ala Leu Lys Lys Met Leu 225 230 235 24020 233 PRT Bacillus 20 Met Ser Arg Tyr Leu Glu Met Leu Ser Leu Phe GlyVal Ala Gly Ala 1 5 10 15 His Pro Gly Gly Leu Ala Phe Ser Lys Ala ValLeu Gln Lys Ala Ala 20 25 30 Pro Ser Pro Asp Gln Pro Ile Leu Asp Ala GlyCys Gly Thr Gly Gln 35 40 45 Thr Ala Ala Tyr Leu Gly His Leu Leu Tyr ProVal Thr Val Val Asp 50 55 60 Lys Asp Pro Ile Met Leu Glu Lys Ala Lys LysArg Phe Ala Asn Glu 65 70 75 80 Gly Leu Ala Ile Pro Ala Tyr Gln Ala GluLeu Glu His Leu Pro Phe 85 90 95 Ser Ser Glu Ser Phe Ser Cys Val Leu SerGlu Ser Val Leu Ser Phe 100 105 110 Ser Arg Leu Thr Ser Ser Leu Gln GluIle Ser Arg Val Leu Lys Pro 115 120 125 Ser Gly Met Leu Ile Gly Ile GluAla Ala Leu Lys Lys Pro Met Pro 130 135 140 Pro Ala Glu Lys Lys Gln MetMet Asp Phe Tyr Gly Phe Thr Cys Leu 145 150 155 160 His Glu Glu Ser GluTrp His Lys Leu Leu Arg Ser Tyr Gly Phe Gln 165 170 175 Lys Thr Glu AlaMet Ser Leu Leu Pro Glu Asp Met Glu Phe Glu Pro 180 185 190 Thr Thr GluMet Asp Leu Ser Gln Thr Ile Asp Pro Ile Tyr Tyr Asp 195 200 205 Thr LeuGln Thr His Tyr Gln Leu Met Gln Leu Tyr Ser Glu Tyr Met 210 215 220 GlyHis Cys Ile Phe Ile Ala Tyr Lys 225 230 21 259 PRT Bacillus 21 Met TrpThr Trp Lys Ala Asp Arg Pro Val Ala Val Ile Val Ile Ile 1 5 10 15 HisGly Ala Ser Glu Tyr His Gly Arg Tyr Lys Trp Leu Ile Glu Met 20 25 30 TrpArg Ser Ser Gly Tyr His Val Val Met Gly Asp Leu Pro Gly Gln 35 40 45 GlyThr Thr Thr Arg Ala Arg Gly His Ile Arg Ser Phe Gln Glu Tyr 50 55 60 IleAsp Glu Val Asp Ala Trp Ile Asp Lys Ala Arg Thr Phe Asp Leu 65 70 75 80Pro Val Phe Leu Leu Gly His Ser Met Gly Gly Leu Val Ala Ile Glu 85 90 95Trp Val Lys Gln Gln Arg Asn Pro Arg Ile Thr Gly Ile Ile Leu Ser 100 105110 Ser Pro Cys Leu Gly Leu Gln Ile Lys Val Asn Lys Ala Leu Asp Leu 115120 125 Ala Ser Lys Gly Leu Asn Val Ile Ala Pro Ser Leu Lys Val Asp Ser130 135 140 Gly Leu Ser Ile Asp Met Ala Thr Arg Asn Glu Asp Val Ile GluAla 145 150 155 160 Asp Gln Asn Asp Ser Leu Tyr Val Arg Lys Val Ser ValArg Trp Tyr 165 170 175 Arg Glu Leu Leu Lys Thr Ile Glu Ser Ala Met ValPro Thr Glu Ala 180 185 190 Phe Leu Lys Val Pro Leu Leu Val Met Gln AlaGly Asp Asp Lys Leu 195 200 205 Val Asp Lys Thr Met Val Ile Lys Trp PheAsn Gly Val Ala Ser His 210 215 220 Asn Lys Ala Tyr Arg Glu Trp Glu GlyLeu Tyr His Glu Ile Phe Asn 225 230 235 240 Glu Pro Glu Arg Glu Asp ValPhe Lys Ala Ala Arg Ala Phe Thr Asp 245 250 255 Gln Tyr Ile

1. A gram positive microorganism comprising an isolated nucleic acidencoding a hydrolase selected from the group consisting of YUXL, YTMA,YITV, YQKD, YCLE, YTAP, YDEN, YBFK, YFHM, YDJP, YVFQ, YVAM, YQJL, SRFAD,YCGS, YTPA, YBAC, YUII, YCLE, YODD, YJCH, YODH.
 2. The gram positivemicroorganism according to claim 1 that is a member of the familyBacillus.
 3. The microorganism according to claim 2 wherein the Bacillusincludes B. subtilis, B. licheniformis, B. lentus, B. brevis, B.stearothermophilus, B. alkalophilus, B. amyloliquefaciens, B. coagulans,B. circulans, B. lautus and B. thuringiensis.
 4. The microorganism ofclaim 3 wherein said microorganism comprises a deletion in a naturallyoccurring protease.
 5. A cleaning composition comprising a hydrolaseselected from the group consisting of YUXL, YTMA, YITV, YQKD, YCLE,YTAP, YDEN, YBFK, YFHM, YDJP, YVFQ, YVAM, YQJL, SRFAD, YCGS, YTPA, YBAC,YUII, YCLE, YODD, YJCH, YODH.
 6. The cleaning composition according toclaim 5 wherein the hydrolase has at least 80% homology with thenaturally occurring hydrolase.
 7. An animal feed comprising a hydrolaseselected from the group consisting of YUXL, YTMA, YITV, YQKD, YCLE,YTAP, YDEN, YBFK, YFHM, YDJP, YVFQ, YVAM, YQJL, SRFAD, YCGS, YTPA, YBAC,YUII, YCLE, YODD, YJCH, YODH.
 8. The animal feed of claim 7 wherein thehydrolase has at least 80% homology with the naturally occurringhydrolase.
 9. A composition for the treatment of a textile comprising ahydrolase selected from the group consisting of YUXL, YTMA, YITV, YQKD,YCLE, YTAP, YDEN, YBFK, YFHM, YDJP, YVFQ, YVAM, YQJL, SRFAD, YCGS, YTPA,YBAC, YUII, YCLE, YODD, YJCH, YODH.
 10. The composition of claim 9wherein the hydrolase has at least 80% homology to the naturallyoccurring hydrolase.
 11. An expression vector comprising a nucleic acidencoding a gram positive hydrolase selected from the group consisting ofYUXL, YTMA, YITV, YQKD, YCLE, YTAP, YDEN, YBFK, YFHM, YDJP, YVFQ, YVAM,YQJL, SRFAD, YCGS, YTPA, YBAC, YUII, YCLE, YODD, YJCH, YODH.
 12. A hostcell comprising an expression vector according to claim
 11. 13. A methodfor detecting a gram positive microorganism hydrolase, comprising thesteps of (a) hybridizing a gram positive microorganism nucleic acid to aprobe, wherein the probe comprises part or all of the naturallyoccurring nucleic acid sequence encoding a hydrolase selected from thegroup consisting of YUXL, YTMA, YITV, YQKD, YCLE, YTAP, YDEN, YBFK,YFHM, YDJP, YVFQ, YVAM, YQJL, SRFAD, YCGS, YTPA, YBAC, YUII, YCLE, YODD,YJCH, YODH; and (b) isolating the gram positive nucleic acid whichhybridizes to said probe.
 14. The method of claim 13, wherein thehybridization takes place under low stringency conditions.